Fructose-4-epimerase and method of producing tagatose using the same

ABSTRACT

Provided are a fructose-4-epimerase variant having tagatose conversion activity, and a method of preparing tagatose using the same.

This application incorporates by reference the computer readablesequence listing in the file “059520_00020_ST25.txt,” created Dec. 9,2020, having 68.2 KB.

TECHNICAL FIELD

The present disclosure relates to a fructose-4-epimerase variant havingimproved conversion activity or stability, and a method of preparingtagatose using the same.

BACKGROUND ART

Tagatose has a natural sweet taste hardly distinguishable from sucroseand also has physical properties similar to sucrose. Tagatose is anatural sweetener, which is present in a small amount in food such asmilk, cheese, cacao, etc., and in sweet fruits such as apples andmandarin. Tagatose has a calorie value of 1.5 kcal/g which is one thirdthat of sucrose, and a glycemic index (GI) of 3 which is 5% that ofsucrose. Tagatose has a sweet taste similar to that of sucrose andvarious health benefits. In this regard, tagatose may be used as analternative sweetener capable of satisfying both health and taste whenapplied to a wide variety of products.

Conventional known methods of producing tagatose include a chemicalmethod (a catalytic reaction) and a biological method (an isomerizingenzyme reaction) of using galactose as a main raw material (see KoreanPatent No. 10-0964091). In order to economically obtain galactose as araw material for the above reactions, studies have been conducted onvarious basic raw materials containing galactose, and a method ofobtaining galactose therefrom to produce tagatose. A representativebasic raw material for obtaining galactose is lactose. However, theprice of lactose or lactose-containing products was unstable, dependingon produced amounts, supply and demand of raw milk and lactose in globalmarkets, etc. Thus, there is a limitation in the stable supply of theraw material for tagatose production. Accordingly, there is a demand fora new method capable of producing tagatose using common saccharides(sucrose, glucose, fructose, etc.).

DISCLOSURE Technical Problem

The present inventors have developed a novel variant protein includingone or more amino acid substitutions in an amino acid sequence of SEQ IDNO: 1, and they found that the variant protein has conversion activityidentical to that of the wild-type of SEQ ID NO: 1, or has improvedconversion activity or stability and improved tagatose productivity, ascompared with the wild-type, thereby completing the present disclosure.

Technical Solution

An object of the present disclosure is to provide a fructose-4-epimerasevariant including one or more amino acid substitutions in an amino acidsequence of SEQ ID NO: 1.

Another object of the present disclosure is to provide a polynucleotideencoding the fructose-4-epimerase variant.

Still another object of the present disclosure is to provide a vectorincluding the polynucleotide.

Still another object of the present disclosure is to provide amicroorganism including the variant.

Still another object of the present disclosure is to provide acomposition for producing tagatose, the composition including one ormore of the fructose-4-epimerase variant; the microorganism expressingthe variant; or a culture of the microorganism.

Still another object of the present disclosure is to provide a method ofpreparing tagatose, the method including the step of reacting fructosein the presence of the microorganism; the culture thereof; or thefructose-4-epimerase derived therefrom.

Advantageous Effects

A fructose-4-epimerase variant of the present disclosure enablesindustrial scale production of tagatose having excellentcharacteristics, and converts fructose, which is a common saccharide,into tagatose, thereby exhibiting a high economical effect.

DESCRIPTION OF DRAWINGS

FIG. 1 shows HPLC chromatography results showing thattagatose-bisphosphate aldolase (CJ_KO_F4E) prepared in one embodiment ofthe present disclosure has fructose-4-epimerase activity; and

FIG. 2 shows a graph of measuring thermal stability of single variantsprepared in one embodiment of the present disclosure at 60° C. overtime.

BEST MODE

The present disclosure will be described in detail as follows.Meanwhile, each description and embodiment disclosed in this disclosuremay also be applied to other descriptions and embodiments. That is, allcombinations of various elements disclosed in this disclosure fallwithin the scope of the present disclosure. Further, the scope of thepresent disclosure is not limited by the specific description describedbelow.

To achieve the objects, one aspect of the present disclosure provides afructose-4-epimerase variant including one or more amino acidsubstitutions in an amino acid sequence of fructose-4-epimerase.

To achieve the objects, another aspect of the present disclosureprovides a fructose-4-epimerase variant including one or more amino acidsubstitutions in an amino acid sequence of SEQ ID NO: 1.

As used herein, the term “fructose-4-epimerase” is an enzyme havingfructose-4-epimerization activity to convert fructose into tagatose byepimerization at C4 position of fructose. With respect to the objects ofthe present disclosure, fructose-4-epimerase may include any enzymewithout limitation, as long as it is able to produce tagatose usingfructose as a substrate, and it may be used interchangeably with‘D-fructose C4-epimerase’. For example, the fructose-4-epimerase mayinclude tagatose bisphosphate aldolase or tagatose-bisphosphate aldolaseclass II accessory protein belonging to EC 4.1.2.40 in a known databaseKEGG (Kyoto Encyclopedia of Genes and Genomes), as long as it hasactivity to convert fructose as a substrate into tagatose. Thetagatose-bisphosphate aldolase is known as an enzyme that producesglycerone phosphate and D-glyceraldehyde 3-phosphate from D-tagatose1,6-bisphosphate as a substrate, as in the following [Reaction Scheme1].D-tagatose 1,6-bisphosphate<=>glycerone phosphate+D-glyceraldehyde3-phosphate  [Reaction Scheme 1]

For example, the fructose-4-epimerase may include tagatose-6-phosphatekinase (EC 2.7.1.144), as long as it has activity to convert fructose asa substrate into tagatose. The tagatose-6-phosphate kinase is known asan enzyme that produces ADP and D-tagatose 1,6-bisphosphate from ATP andD-tagatose 6-phosphate as a substrate, as in the following [ReactionScheme 2].ATP+D-tagatose 6-phosphate<=>ADP+D-tagatose 1,6-bisphosphate  [ReactionScheme 2]

The activity of fructose-4-epimerase may have a conversion rate oftagatose from fructose as a substrate (conversion rate=tagatoseweight/initial fructose weight*100) of 0.01% or more, specifically 0.1%or more, and more specifically 0.3% or more. Much more specifically, theconversion rate may be in the range of 0.01% to 100% or in the range of0.1% to 50%.

The fructose-4-epimerase, tagatose-bisphosphate aldolase, ortagatose-6-phosphate kinase of the present disclosure may be an enzymederived from a heat-resistant microorganism or a variant thereof, forexample, an enzyme derived from Kosmotoga olearia, Thermanaerothrixdaxensis, Rhodothermus profundi, Rhodothermus marinus, Limnochordapilosa, Caldithrix abyssi, Caldilinea aerophila, Thermoanaerobacterthermohydrosulfuricus, Acidobacteriales bacterium, Caldicellulosiruptorkronotskyensis, Thermoanaerobacterium thermosaccharolyticum, orPseudoalteromonas sp. H103, or a variant thereof, but is not limitedthereto, specifically, an enzyme derived from Kosmotoga olearia (SEQ IDNO: 1), Thermoanaerobacterium thermosaccharolyticum (SEQ ID NO: 3),Pseudoalteromonas sp. H103 (SEQ ID NO: 5), Thermanaerothrix daxensis(SEQ ID NO: 7), Acidobacteriales bacterium (SEQ ID NO: 9), Rhodothermusprofundi (SEQ ID NO: 11), Rhodothermus marinus (SEQ ID NO: 13),Limnochorda pilosa (SEQ ID NO: 15), Caldithrix abyssi (SEQ ID NO: 17),Caldicellulosiruptor kronotskyensis (SEQ ID NO: 19), Caldilineaaerophila (SEQ ID NO: 21), or Thermoanaerobacter thermohydrosulfuricus(SEQ ID NO: 23), or a variant thereof, but is not limited thereto.

Specifically, the fructose-4-epimerase, tagatose-bisphosphate aldolase,or tagatose-6-phosphate kinase may include an amino acid sequence of SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, or an amino acidsequence having 70% or higher homology or identity thereto, but is notlimited thereto. More specifically, the fructose-4-epimerase of thepresent disclosure may include a polypeptide having at least 60%, 70%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to theamino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,or 23. Further, it is apparent that an accessory protein having an aminoacid sequence having the homology or identity and exhibiting theefficacy corresponding to the above protein is also included in thescope of the present disclosure, although a partial sequence of theamino acid sequence is deleted, modified, substituted, or added.

In the present disclosure, SEQ ID NO: 1 means an amino acid sequencehaving fructose-4-epimerase activity. The sequence of SEQ ID NO: 1 maybe obtained from a known database, GenBank of NCBI or KEGG (KyotoEncyclopedia of Genes and Genomes). For example, the sequence may bederived from Kosmotoga olearia, more specifically, a polypeptide/proteinincluding the amino acid sequence of SEQ ID NO: 1, but is not limitedthereto. Further, a sequence having activity identical to the aboveamino acid sequence may be included without limitation. Further, theamino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70%or higher homology or identity thereto may be included, but is notlimited thereto. Specifically, the amino acid sequence may include theamino acid sequence having SEQ ID NO: 1 and an amino acid sequencehaving at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higherhomology or identity to SEQ ID NO: 1. Further, it is apparent that aprotein having an amino acid sequence having the homology or identityand exhibiting the efficacy corresponding to the above protein is alsoincluded in the scope of the present disclosure, although a partialsequence of the amino acid sequence is deleted, modified, substituted,or added.

That is, although described as “a protein having an amino acid sequenceof a particular SEQ ID NO” in the present disclosure, the protein mayhave an activity that is identical or similar to that of a proteinconsisting of an amino acid sequence of the corresponding SEQ ID NO. Insuch a case, it is obvious that any proteins having an amino acidsequence with deletion, modification, substitution, conservativesubstitution, or addition in part of the sequence also can be used inthe present disclosure. For example, in the case of having the activitythat is the same as or corresponding to that of the modified protein, itdoes not exclude an addition of a sequence upstream or downstream of theamino acid sequence, which does not alter the function of the protein, amutation that may occur naturally, a silent mutation thereof, or aconservative constitution, and even when the sequence addition ormutation is present, it obviously belongs to the scope of the presentdisclosure.

As used herein, the term “tagatose” is, a kind of ketohexose which is amonosaccharide, used interchangeably with “D-tagatose”

As used herein, the term “fructose-4-epimerase variant” means afructose-4-epimerase variant including one or more amino acidsubstitutions in the amino acid sequence of the polypeptide havingfructose-4-epimerase activity.

Specifically, the amino acid substitution includes i) substitution ofanother amino acid for one or more selected from the group consisting ofamino acids at positions 52, 136, 197, 317, and 320, or ii) substitutionof glutamic acid (E) for an amino acid at position 414 from theN-terminus.

As used herein, ‘position N’ may include position N and an amino acidposition corresponding to the position N, specifically, an amino acidposition corresponding to any amino acid residue in a mature polypeptidedisclosed in a particular amino acid sequence. The particular amino acidsequence may be any one of the amino acid sequences of SEQ ID NOS: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, and 23.

The amino acid position corresponding to the position N or the aminoacid position corresponding to any amino acid residue in the maturepolypeptide disclosed in the particular amino acid sequence may bedetermined using the Needleman-Wunsch algorithm (literature [Needlemanand Wunsch, 1970, J. Mol. Biol. 48: 443-453]), specifically, version5.0.0 or later, as implemented in the Needle program of the EMBOSSpackage (EMBOSS: The European Molecular Biology Open Software Suite,literature [Rice et al., 2000, Trends Genet. 16:276-277]). Parametersused may be gap open penalty of 10, gap extension penalty of 0.5, andEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the amino acid residue at the amino acid positioncorresponding to the position N or at the amino acid positioncorresponding to any amino acid residue in the mature polypeptidedisclosed in the particular amino acid sequence may be determined byalignment of multiple polypeptide sequences using several computerprograms including, but not limited to, MUSCLE (multiple sequencecomparison by log-expectation; version 3.5 or later; literature [Edgar,2004, Nucleic Acids Research 32: 1792-1797]), MAFFT (version 6.857 orlater; literature [Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066]; literature [Katoh et al., 2005, Nucleic Acids Research 33:511-518]; literature [Katoh and Toh, 2007, Bioinformatics 23: 372-374];literature [Katoh et al., 2009, Methods in Molecular Biology 537:39-64]; literature [Katoh and Toh, 2010, Bioinformatics 26: 1899-1900]),and EMBOSS EMMA employing ClustalW (1.83 or later; literature [Thompsonet al., 1994, Nucleic Acids Research 22: 4673-4680]), using theirrespective default parameters.

When the other polypeptide has diverged from the mature polypeptide ofthe particular amino acid sequence such that traditional sequence-basedcomparison fails to detect their relationship (literature [Lindahl andElofsson, 2000, J. Mol. Biol. 295: 613-615]), other pairwise sequencecomparison algorithms may be used. Greater sensitivity in sequence-basedsearching may be attained using search programs that utilizeprobabilistic representations of polypeptide families (profiles) tosearch databases. For example, PSI-BLAST program generates profilesthrough an iterative database search process and is capable of detectingremote homologs (literature [Atschul et al., 1997, Nucleic Acids Res.25: 3389-3402]). Even greater sensitivity may be achieved if the familyor superfamily for the polypeptide has one or more representatives inthe protein structure databases. Programs such as GenTHREADER(literature [Jones, 1999, J. Mol. Biol. 287: 797-815]; literature[McGuffin and Jones, 2003, Bioinformatics 19: 874-881]) utilizeinformation from a variety of sources (PSI-BLAST, secondary structureprediction, structural alignment profiles, and solvation potentials) asinput to a neural network that predicts the structural folding for aquery sequence. Similarly, the method of literature [Gough et al., 2000,J. Mol. Biol. 313: 903-919] may be used to align a sequence of unknownstructure with the superfamily models present in the SCOP database.These alignments may in turn be used to generate homology, similarity,or identity models for the polypeptide, and such models may be assessedfor accuracy using a variety of tools developed for that purpose.

The ‘another polypeptide’ of i) is not limited, as long as it is anamino acid other than the amino acid corresponding to the position.‘Amino acids’ are classified into four types of acidic, basic, polar(hydrophilic), and nonpolar (hydrophobic) amino acids according toproperties of their side chains.

The variant may be a protein having substitution of one or more aminoacids selected from the group consisting of nonpolar amino acidsincluding glycine (G), alanine (A), valine (V), leucine (L), isoleucine(I), methionine (M), phenylalanine (F), tryptophan (W), and proline (P);polar amino acids including serine (S), threonine (T), cysteine (C),tyrosine (Y), aspartic acid (D), and glutamine (Q); acidic amino acidsincluding asparagine (N) and glutamic acid (E); and basic amino acidsincluding lysine (K), arginine (R), and histidine (H) for an amino acidat each position of the amino acid sequence of SEQ ID NO: 1, but is notlimited thereto.

Specifically, the amino acid at position 52 may be substituted by anonpolar amino acid, or a polar amino acid, more specifically,methionine (M), serine (S), threonine (T), or leucine (L). The aminoacid at position 136 may be substituted by a nonpolar amino acid, or apolar amino acid, more specifically, phenylalanine (F), tryptophan (W),proline (P), or tyrosine (Y). The amino acid at position 197 may besubstituted by a nonpolar amino acid or a polar amino acid, morespecifically, alanine (A) or serine (S). The amino acid at position 317may be substituted by a nonpolar amino acid or polar amino acid, morespecifically, phenylalanine (F) or tyrosine (Y).

The fructose-4-epimerase variant may include a polypeptide, of which oneor more amino acids differ from the recited sequence in conservativesubstitutions and/or modifications, in addition to substitution ofanother amino acid for the amino acid at the particular position, whileretaining functions or properties of the protein.

As used herein, the term “conservative substitution” means substitutionof one amino acid with another amino acid that has similar structuraland/or chemical properties. The variant may have, for example, one ormore conservative substitutions while retaining one or more biologicalactivities. The conservative substitution has little or no impact on theactivity of a resulting polypeptide.

Further, variants having variation of one or more amino acids inaddition to the amino acids at the above-described particular positionsmay include deletion or addition of amino acids that have minimalinfluence on properties and a secondary structure of the polypeptide.For example, a polypeptide may be conjugated to a signal (or leader)sequence at the N-terminus of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to other sequence or a linker for identification,purification, or synthesis of the polypeptide.

Further, the variant includes the above-described variations of SEQ IDNO: 1 and/or amino acids having at least 70%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% or higher homology or identity to SEQ ID NO: 1 otherthan the variations and positions of SEQ ID NO: 1. The variations of SEQID NO: 1 are as described above, and homology or identity thereto may behomology or identity at positions other than the above-describedvariations.

With respect to the objects of the present disclosure, thefructose-4-epimerase variant is characterized by having improvedstability, as compared with the wild-type.

The term “stability” means having thermal stability of an enzyme havinghigh heat resistance.

Specifically, the fructose-4-epimerase variant of SEQ ID NO: 1 ischaracterized in that its thermal stability is improved, as comparedwith the wild-type of SEQ ID NO: 1.

For example, the fructose-4-epimerase variant of the present disclosuremay be an enzyme having high heat resistance. Specifically, thefructose-4-epimerase variant of the present disclosure may exhibit 50%to 100%, 60% to 100%, 70% to 100%, or 75% to 100% activity of themaximum activity at 50° C. to 70° C. More specifically, thefructose-4-epimerase variant of the present disclosure may exhibit 80%to 100% or 85% to 100% activity of the maximum activity at 55° C. to 60°C., 60° C. to 70° C., 55° C., 60° C., or 70° C.

Examples of the fructose-4-epimerase variant may include those describedin Tables 3 to 5, but are not limited thereto.

Another aspect of the present disclosure provides a polynucleotideencoding the fructose-4-epimerase variant, or a vector including thepolynucleotide.

As used herein, the term “polynucleotide” refers to a DNA or RNA strandhaving a predetermined length or more, which is a long chain polymer ofnucleotides formed by linking nucleotide monomers via covalent bonds.More specifically, the polynucleotide refers to a polynucleotidefragment encoding the variant protein.

The polynucleotide encoding the fructose-4-epimerase variant of thepresent disclosure may include any polynucleotide sequence withoutlimitation, as long as it is a polynucleotide sequence encoding thefructose-4-epimerase variant of the present disclosure. For example, thepolynucleotide encoding the fructose-4-epimerase variant of the presentdisclosure may be a polynucleotide sequence encoding the amino acidsequence, but is not limited thereto. In the polynucleotide, variousmodifications may be made in the coding region provided that they do notchange the amino acid sequence of the protein, due to codon degeneracyor in consideration of the codons preferred by the organism in which theprotein is to be expressed. Therefore, it is apparent that, due to codondegeneracy, a polynucleotide which may be translated into thepolypeptide composed of the amino acid sequence or the polypeptidehaving homology or identity thereto may also be included.

Further, a probe which may be produced from a known nucleotide sequence,for example, a sequence which hybridizes with a complementary sequenceto all or a part of the nucleotide sequence under stringent conditionsto encode the fructose-4-epimerase variant may also be included withoutlimitation.

The term “stringent conditions” mean conditions under which specifichybridization between polynucleotides is allowed. Such conditions aredescribed in detail in a literature (e.g., J. Sambrook et al., supra).For example, the stringent conditions may include, for example,conditions under which genes having high homology or identity, 70% orhigher, 80% or higher, 85% or higher, specifically 90% or higher, morespecifically 95% or higher, much more specifically 97% or higher,particularly specifically 99% or higher homology or identity arehybridized with each other and genes having homology or identity lowerthan the above homology or identity are not hybridized with each other,or ordinary washing conditions of Southern hybridization, i.e., washingonce, specifically, twice or three times at a salt concentration and atemperature corresponding to 60° C., 1×SSC, 0.1% SDS, specifically, 60°C., 0.1×SSC, 0.1% SDS, and more specifically 68° C., 0.1×SSC, 0.1% SDS.

Although a mismatch between nucleotides may occur due to the stringencyof hybridization, it is required that the two nucleic acids have acomplementary sequence. The term “complementary” is used to describe therelationship between nucleotide bases which may hybridize with eachother. For example, with regard to DNA, adenosine is complementary tothymine and cytosine is complementary to guanine. Accordingly, thepresent disclosure may include not only the substantially similarnucleic acid sequences but also isolated nucleic acid fragments whichare complementary to the entire sequence.

Specifically, the polynucleotide having homology or identity may bedetected using hybridization conditions including the hybridization stepat a Tm value of 55° C. and the conditions described above.Additionally, the Tm value may be 60° C., 63° C., or 65° C., but is notlimited thereto, and may be appropriately controlled by one of ordinaryskill in the art according to the purposes.

Appropriate stringency for the hybridization of polynucleotides dependson the length and degree of complementarity of the polynucleotides, andthe variables are well-known in the art (see Sambrook et al., supra,9.50-9.51, 11.7-11.8).

As used herein, the term ‘homology’ or ‘identity’ means the degree ofrelevance between two given amino acid sequences or nucleotidesequences, and may be expressed as a percentage.

The terms ‘homology’ and ‘identity’ may be often used interchangeably.

The sequence homology or identity of the conserved polynucleotide orpolypeptide may be determined by standard alignment algorithms, and maybe used with default gap penalties established by the used program.Substantially, homologous or identical sequences may hybridize undermoderately or highly stringent conditions such that the full length ofthe sequence or at least about 50%, 60%, 70%, 80%, or 90% or more of thefull-length may hybridize. Also, contemplated are polynucleotides thatcontain degenerate codons in place of codons in the hybridization.

Whether or not any two polynucleotide or polypeptide sequences havehomology, similarity, or identity may be determined using known computeralgorithms such as the “FASTA” program, using, for example, the defaultparameters as in Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]:2444], or determined using the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleprogram of the EMBOSS package (EMBOSS: The European Molecular BiologyOpen Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277)(version 5.0.0 or later) (including GCG program package (Devereux, J.,et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA(Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide toHuge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994, and [CARILLO ETA/.](1988) SIAM J Applied Math 48: 1073). Forexample, BLAST of the National Center for Biotechnology Informationdatabase, or ClustalW may be used to determine homology, similarity, oridentity.

Homology, similarity, or identity of polynucleotides or polypeptides maybe determined, for example, by comparing sequence information using aGAP computer program such as Needleman et al. (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482.Briefly, the GAP program defines similarity as the number of alignedsymbols (i.e., nucleotides or amino acids), which are similar, dividedby the total number of symbols in the shorter of the two sequences.Default parameters for the GAP program may include: (1) a binarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) and the weighted comparison matrix of Gribskov et al(1986) Nucl. Acids Res. 14: 6745, as disclosed in Schwartz and Dayhoff,eds., Atlas Of Protein Sequence And Structure, National BiomedicalResearch Foundation, pp. 353-358 (1979) (or EDNAFULL (EMBOSS version ofNCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap andan additional 0.10 penalty for each symbol in each gap (or gap openpenalty of 10, gap extension penalty of 0.5); and (3) no penalty for endgaps. Therefore, as used herein, the term “homology” or “identity”represents relevance between sequences.

As used herein, the term “vector” means a DNA construct that includes anucleotide sequence of a polynucleotide encoding a target variantprotein operably linked to an appropriate regulatory sequence to enableexpression of the target variant protein in an appropriate host cell.The regulatory sequence may include a promoter capable of initiatingtranscription, any operator sequence for the regulation of suchtranscription, a sequence of an appropriate mRNA ribosome-bindingdomain, and a sequence regulating termination of transcription andtranslation. After the vector is transformed into the appropriate hostcell, it may replicate or function independently of the host genome, andmay be integrated into the genome itself.

The vector used in the present disclosure is not particularly limited,as long as it is able to replicate in the host cell, and any vectorknown in the art may be used. Examples of commonly used vectors mayinclude a natural or recombinant plasmid, cosmid, virus, andbacteriophage. For instance, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII,t10, t11, Charon4A, Charon21A, etc. may be used as a phage vector orcosmid vector. As a plasmid vector, pBR type, pUC type, pBluescriptIItype, pGEM type, pTZ type, pCL type, pET type, etc. may be used.Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322,pMW118, pCC1BAC vector, etc. may be used.

For example, a polynucleotide encoding a target variant protein in thechromosome may be replaced by a mutated polynucleotide using a vectorfor intracellular chromosomal insertion. The chromosomal insertion ofthe polynucleotide may be performed by any method known in the art, forexample, homologous recombination, but is not limited thereto. Aselection marker to confirm the chromosomal insertion may be furtherincluded. The selection marker is to select cells transformed with thevector, that is, to confirm insertion of the desired nucleotidemolecule, and the selection marker may include markers providingselectable phenotypes, such as drug resistance, auxotrophy, resistanceto cytotoxic agents, or expression of surface-modified proteins. Sinceonly cells expressing the selection marker are able to survive or toshow different phenotypes under the environment treated with a selectiveagent, the transformed cells may be selected.

As still another aspect of the present disclosure, the presentdisclosure provides a microorganism producing tagatose, themicroorganism including the variant protein or the polynucleotideencoding the variant protein. Specifically, the microorganism includingthe variant protein and/or the polynucleotide encoding the variantprotein may be a microorganism prepared by transforming with the vectorincluding the polynucleotide encoding the variant protein, but is notlimited thereto.

As used herein, the term “transformation” means introduction of a vectorincluding a polynucleotide encoding a target protein into a host cell insuch a way that the protein encoded by the polynucleotide is expressedin the host cell. As long as the transformed polynucleotide may beexpressed in the host cell, it may be integrated into and placed in thechromosome of the host cell, or it may exist extrachromosomally, orirrespective thereof. Further, the polynucleotide includes DNA and RNAencoding the target protein. The polynucleotide may be introduced in anyform, as long as it may be introduced into the host cell and expressedtherein. For example, the polynucleotide may be introduced into the hostcell in the form of an expression cassette, which is a gene constructincluding all elements required for its autonomous expression. Commonly,the expression cassette includes a promoter operably linked to thepolynucleotide, transcriptional termination signals, ribosome bindingsites, and translation termination signals. The expression cassette maybe in the form of a self-replicable expression vector. Also, thepolynucleotide as it is may be introduced into the host cell andoperably linked to sequences required for expression in the host cell,but is not limited thereto.

As used herein, the term “operably linked” means a functional linkagebetween a promoter sequence which initiates and mediates transcriptionof the polynucleotide encoding the target variant protein of the presentdisclosure and the polynucleotide sequence.

Still another aspect of the present disclosure provides a microorganismproducing fructose-4-epimerase, the microorganism including thefructose-4-epimerase variant, the polynucleotide encoding thefructose-4-epimerase variant, or the vector including thepolynucleotide.

As used herein, the term “microorganism including thefructose-4-epimerase variant” may refers to a recombinant microorganismto express the fructose-4-epimerase variant of the present disclosure.For example, the microorganism refers to a host cell or a microorganismwhich is able to express the variant by including the polynucleotideencoding the fructose-4-epimerase variant or by transforming with thevector including the polynucleotide encoding the fructose-4-epimerasevariant. With respect to the objects of the present disclosure, themicroorganism is specifically a microorganism expressing thefructose-4-epimerase variant including one or more amino acidsubstitutions in the amino acid sequence of SEQ ID NO: 1, and themicroorganism may be a microorganism expressing the variant proteinhaving the fructose-4-epimerase activity, wherein the amino acidsubstitution is substitution of one or more amino acids at one or morepositions from the N-terminus, but is not limited thereto.

The fructose-4-epimerase variant of the present disclosure may beobtained by transforming a microorganism such as E. coli with DNAexpressing the enzyme of the present disclosure or the variant thereof,culturing the microorganism to obtain a culture, disrupting the culture,and then performing purification using a column, etc. The microorganismfor transformation may include Corynebacterium glutamicum, Aspergillusoryzae, or Bacillus subtilis, in addition to Escherichia coli, but isnot limited thereto.

The microorganism of the present disclosure may include either aprokaryotic microorganism or a eukaryotic microorganism, as long as itis a microorganism capable of producing the fructose-4-epimerase of thepresent disclosure by including the nucleic acid of the presentdisclosure or the recombinant vector of the present disclosure. Forexample, the microorganism may include microorganism strains belongingto the genus Escherichia, the genus Erwinia, the genus Serratia, thegenus Providencia, the genus Corynebacterium, and the genusBrevibacterium, but is not limited thereto.

The microorganism of the present disclosure may include anymicroorganism capable of expressing the fructose-4-epimerase of thepresent disclosure by various known methods, in addition to introductionof the nucleic acid or the vector.

The culture of the microorganism of the present disclosure may beproduced by culturing, in a medium, the microorganism capable ofexpressing the fructose-4-epimerase of the present disclosure.

In the method, the “culturing” means that the microorganism is allowedto grow under appropriately controlled environmental conditions. Thestep of culturing the microorganism may be, but is not particularlylimited to, carried out by a known batch culture method, continuousculture method, or fed batch culture method. With regard to the cultureconditions, a proper pH (e.g., pH 5 to 9, specifically pH 6 to 8, andmost specifically pH 6.8) may be adjusted using a basic compound (e.g.,sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound(e.g., phosphoric acid or sulfuric acid), but is not particularlylimited thereto. Oxygen or an oxygen-containing gas mixture may beinjected into the culture to maintain aerobic conditions. The culturetemperature may be maintained from 20° C. to 45° C., and specifically,from 25° C. to 40° C. for about 10 hours to about 160 hours, but is notlimited thereto.

Furthermore, the culture medium to be used may include, as carbonsources, sugars and carbohydrates (e.g., glucose, sucrose, lactose,fructose, maltose, molasse, starch, and cellulose), oil and fat (e.g.,soybean oil, sunflower seed oil, peanut oil, and coconut oil), fattyacids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols(e.g., glycerol and ethanol), and organic acids (e.g., acetic acid)individually or in combination, but is not limited thereto. As nitrogensources, nitrogen-containing organic compounds (e.g., peptone, yeastextract, meat broth, malt extract, corn steep liquor, soybean meal, andurea), or inorganic compounds (e.g., ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate)may be used individually or in combination, but are not limited thereto.As phosphorus sources, dipotassium hydrogen phosphate, potassiumdihydrogen phosphate, and corresponding sodium salts thereof may be usedindividually or in combination, but are not limited thereto. Further,the medium may include essential growth-stimulating substances includingother metal salts (e.g., magnesium sulfate or iron sulfate), aminoacids, and vitamins.

Still another aspect of the present disclosure provides a compositionfor producing tagatose, the composition including thefructose-4-epimerase variant; the microorganism expressing the same; orthe culture of the microorganism.

The composition for producing tagatose of the present disclosure mayfurther include fructose.

In addition, the composition for producing tagatose of the presentdisclosure may further include any appropriate excipient commonly usedin the corresponding composition for producing tagatose. The excipientmay include, for example, a preservative, a wetting agent, a dispersingagent, a suspending agent, a buffer, a stabilizer, an isotonic agent,etc., but is not limited thereto.

The composition for producing tagatose of the present disclosure mayfurther include a metal ion or a metal salt. In a specific embodiment, ametal of the metal ion or the metal salt may be a metal containing adivalent cation. Specifically, the metal of the present disclosure maybe nickel (Ni), iron (Fe), cobalt (Co), magnesium (Mg), or manganese(Mn). More specifically, the metal salt may be MgSO₄, FeSO₄, NiSO₄,NiCl₂, MgCl₂, CoSO₄, MnCl₂, or MnSO₄.

Still another aspect of the present disclosure provides a method ofpreparing tagatose, the method including the step of converting fructoseinto tagatose by contacting fructose with the fructose-4-epimerasevariant; the microorganism including the fructose-4-epimerase variant;or the culture thereof, specifically, a method of preparing tagatosefrom fructose using the fructose-4-epimerase variant as afructose-4-epimerase.

For example, the contacting of the present disclosure may be performedunder a condition of pH 5.0 to pH 9.0, a temperature condition of 30° C.to 80° C., and/or for 0.5 hr to 48 hr.

Specifically, the contacting of the present disclosure may be performedunder a condition of pH 6.0 to pH 9.0 or pH 7.0 to pH 9.0. Further, thecontacting of the present disclosure may be performed under atemperature condition of 35° C. to 80° C., 40° C. to 80° C., 45° C. to80° C., 50° C. to 80° C., 55° C. to 80° C., 60° C. to 80° C., 30° C. to70° C., 35° C. to 70° C., 40° C. to 70° C., 45° C. to 70° C., 50° C. to70° C., 55° C. to 70° C., 60° C. to 70° C., 30° C. to 65° C., 35° C. to65° C., 40° C. to 65° C., 45° C. to 65° C., 50° C. to 65° C., 55° C. to65° C., 30° C. to 60° C., 35° C. to 60° C., 40° C. to 60° C., 45° C. to60° C., 50° C. to 60° C. or 55° C. to 60° C. Further, the contacting ofthe present disclosure may be performed for 0.5 hr to 36 hr, 0.5 hr to24 hr, 0.5 hr to 12 hr, 0.5 hr to 6 hr, 1 hr to 48 hr, 1 hr to 36 hr, 1hr to 24 hr, 1 hr to 12 hr, 1 hr to 6 hr, 3 hr to 48 hr, 3 hr to 36 hr,3 hr to 24 hr, 3 hr to 12 hr, 3 hr to 6 hr, 6 hr to 48 hr, 6 hr to 36hr, 6 hr to 24 hr, 6 hr to 12 hr, 12 hr to 48 hr, 12 hr to 36 hr, 12 hrto 24 hr, 18 hr to 48 hr, 18 hr to 36 hr, or 18 hr to 30 hr.

Further, the contacting of the present disclosure may be performed inthe presence of a metal ion or a metal salt. The applicable metal ion ormetal salt is the same as described above.

The production method of the present disclosure may further include thestep of separating and/or purifying the produced tagatose. Theseparation and/or purification may be performed using a method commonlyused in the art. Non-limiting examples may include dialysis,precipitation, adsorption, electrophoresis, ion exchange chromatography,fractional crystallization, etc. The purification may be performed onlyby a single method or by two or more methods in combination.

In addition, the production method of the present disclosure may furtherinclude the step of performing decolorization and/or deionization,before or after the separation and/or purification step(s). Byperforming the decolorization and/or deionization, it is possible toobtain tagatose with higher quality.

For another example, the production method of the present disclosure mayfurther include the step of performing crystallization of tagatose,after the step of converting into tagatose of the present disclosure,performing the separation and/or purification, or performing thedecolorization and/or deionization. The crystallization may be performedby a crystallization method commonly used. For example, thecrystallization may be performed by cooling crystallization.

Further, the production method of the present disclosure may furtherinclude the step of concentrating tagatose, before the step ofperforming crystallization. The concentrating may increase thecrystallization efficiency.

For another example, the production method of the present disclosure mayfurther include the step of contacting unreacted fructose with theenzyme of the present disclosure, the microorganism expressing theenzyme, or the culture of the microorganism after the step of separationand/or purification, or the step of reusing a crystal-separated mothersolution in the step of separation and/or purification after the step ofperforming the crystallization of the present disclosure, or acombination thereof. The additional steps are economically advantageousin that tagatose may be obtained with higher yield and the amount offructose to be discarded may be reduced.

MODE FOR INVENTION

Hereinafter, the present disclosure will be described in more detailwith reference to Examples. However, these Examples are for the purposeof illustrating the present disclosure, and the scope of the presentdisclosure is not intended to be limited by these Examples. It will beapparent to those skilled in the art to which the present disclosurepertains.

Example 1. Preparation of Recombinant Expression Vectors andTransformants, Each Including Wild-Type Fructose-4-Epimerase Gene orImproved Fructose-4-Epimerase Gene Example 1-1. Preparation ofRecombinant Expression Vectors Including Fructose-4-Epimerase Wild-TypeGene

To prepare fructose-4-epimerase, genetic information of Kosmotogaolearia-derived fructose-4-epimerase was obtained to prepare a vectorexpressible in E. coli and a transformed microorganism (transformant).It was confirmed that the sequence may be used as a fructose-4-epimeraseto convert fructose into tagatose (FIG. 1 ).

In detail, a nucleotide sequence of fructose-4-epimerase was selectedfrom nucleotide sequences of Kosmotoga olearia, which is registered inKEGG (Kyoto Encyclopedia of Genes and Genomes). Based on the informationof the amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQID NO: 2) of Kosmotoga olearia, it was inserted into pBT7-C-His which isa vector expressible in E. coli to synthesize and prepare a recombinantexpression vector pBT7-C-His-KO, performed by Bioneer Corp.

Example 1-2. Preparation of Improved Fructose-4-Epimerase Library andScreening of Activity-Improved Variant

Random mutation was performed using Kosmotoga olearia-derivedfructose-4-epimerase gene as a template to construct afructose-4-epimerase variant library. In detail, random mutation wasinduced using a diversify random mutagenesis kit (ClonTech) to generate2 to 3 variations per 1000 base pairs in the fructose-4-epimerase gene.PCR reaction conditions are shown in the following Tables 1 and 2. Thegene library encoding the fructose-4-epimerase variant was constructedand inserted into E. coli BL21(DE3).

TABLE 1 Composition of reaction solution Addition amount (μl) PCR GradeWater 36 10X TITANIUM Taq Buffer 5 MnSO4 (8 mM) 4 dGTP (2 mM) 1 50XDiversify dNTP Mix 1 Primer mix 1 Template DNA 1 TITANIUM Taq Polym. 1

TABLE 2 Step Temperature (° C.) Time (sec) Cycle Initial Denaturation 9430 1 Denaturation 94 30 25 Annealing/Extension 68 60 Final Extension 6860 1 soak 4 —

E. coli BL21(DE3) having the pBT7-C-His plasmid harboring thefructose-4-epimerase variant gene was seeded in a deep well rackcontaining 0.2 mL of an LB liquid medium supplemented with an ampicillinantibiotic, and seed-cultured in a shaking incubator at 37° C. for 16hours or longer. The culture broth obtained from the seed culture wasseeded in a culture deep well rack containing a liquid medium containingLB and lactose which is a protein expression regulator, followed by mainculture. The seed culture and main culture were performed underconditions of a shaking speed of 180 rpm and 37° C. Next, the culturebroth was centrifuged at 4,000 rpm and 4° C. for 20 minutes, and thenthe microorganism was recovered and subjected to an activity test.

For high-speed screening of a large amount of the activity-improvedvariant enzyme from the prepared random mutation library, a colorimetricmethod capable of specifically quantifying D-fructose was used. Indetail, a 70% folin-ciocalteu reagent (SIGMA-ALDRICH) and a substratereaction solution were mixed at a ratio of 15:1, and allowed to react at80° C. for 5 minutes. OD values were measured at 900 nm to selectvariants having the activity (conversion of D-fructose into D-tagatose)by comparing the relative activity thereof with that of the wild-typeenzyme (SEQ ID NO: 1). 10 colonies thus selected were sequenced toexamine their base sequences. As a result, a total of 6 positions (52,136, 197, 317, 320, and 414) were found to be mutated.

Example 2. Preparation of Variant Enzymes and Selection ofStability-Improved Variant Enzymes

A single-site saturation mutagenesis library of 6 target positions (52,136, 197, 317, 320, and 414) selected in Example 1-2 was constructed toprepare variant enzymes, and stability-improved variation sites andamino acids were screened and selected. Information of the improvedsites as selected above was incorporated to prepare variant enzymes, andvariant enzymes having improved stability of thefructose-4-epimerization conversion reaction were developed.

Example 2-1. Saturation Mutagenesis

The recombinant expression vector pBT7-C-His-KO which was prepared forexpressing the wild-type enzyme gene in E. coli BL21(DE3) (expressingthe recombinant enzyme having 6×His-tag at the C-terminus of thewild-type) was used as a template for saturation mutagenesis for theconstruction of a variant library. In view of mutation frequencyvariation and variant yield, etc., inversed PCR-based saturationmutagenesis was used (2014. Anal. Biochem. 449:90-98), and in order tominimize screening scales of the constructed variant library (minimizethe number of codons introduced for saturation mutagenesis), a mixedprimer NDTNMA/ATG/TGG (2012. Biotechniques 52:149-158) in which stopcodons were excluded and rare codons for E. coli were minimized wasdesigned and used. In detail, a primer having a total length of 33 bpwas constructed using 15 bp residing at the front side, 3 bp to besubstituted, and 15 bp residing at the rear side of each site. PCR wasperformed by repeating 30 cycles consisting of denaturing at 94° C. for2 minutes, denaturing at 94° C. from 30 seconds, annealing at 60° C. for30 seconds, and extending at 72° C. for 10 minutes, followed byelongation at 72° C. for 60 minutes. After construction of a saturationmutagenesis library for the selected amino acid sites, variants for eachlibrary were randomly selected (<11 variations). Base sequences wereanalyzed to evaluate amino acid mutation frequency. Based on theanalysis results, scales of screening each library were set withsequence coverage of 90% or more (2003. Nucleic Acids Res. 15; 31:e30).

Through the saturation mutagenesis, variant candidates retainingexcellent characteristics were prepared, and sequencing analysis wasperformed to examine the variation sites. Thus, a total of 11 variantswere obtained (Table 3).

TABLE 3 Variation site D136 F P W Y L320 F C52 M T S L V197 A S T317 Y FS414 E

Example 2-2. Preparation of Stability-Improved Variant Enzymes

In order to evaluate relative activity of fructose-4-epimerization for avariant enzyme at a single site with improved stability and a variantenzyme at a single site with combination thereof, the saturationmutagenesis library gene prepared in 3-1 was transformed into E. coliBL21(DE3), and each transformed microorganism was seeded in a culturetube containing 5 mL of LB liquid medium containing an ampicillinantibiotic, and seed-cultured in a shaking incubator at 37° C. untilabsorbance at 600 nm reached 2.0. The culture broth obtained from theseed culture was seeded in a culture flask containing a liquid mediumcontaining LB and lactose which is a protein expression regulator,followed by main culture. The seed culture and main culture wereperformed under conditions of a shaking speed of 180 rpm and 37° C.Next, the culture broth was centrifuged at 8,000 rpm and 4° C. for 20minutes, and then the microorganism was recovered. The recoveredmicroorganism was washed with a 50 mM Tris-HCl (pH 8.0) buffer solutiontwice, and resuspended in a 50 mM NaH₂PO₄ (pH 8.0) buffer solutioncontaining 10 mM imidazole and 300 mM NaCl. The resuspendedmicroorganism was disrupted using a sonicator, and centrifuged at 13,000rpm and 4° C. for 20 minutes to collect only the supernatant. Thesupernatant was purified using His-taq affinity chromatography, and a 50mM NaH₂PO₄ (pH 8.0) buffer solution containing 20 mM imidazole and 300mM NaCl was applied in a 10-fold volume of a filler to removenon-specific binding proteins. Subsequently, 50 mM NaH₂PO₄ (pH 8.0)buffer solution containing 250 mM imidazole and 300 mM NaCl was furtherapplied to perform elution and purification. Then, dialysis wasperformed using a 50 mM Tris-HCl (pH 8.0) buffer solution, and therespective purified enzymes were obtained for characterization of theenzymes.

Example 3. Comparative Evaluation of Characteristics ofStability-Improved Variant Enzymes

To measure the fructose-4-epimerization activity of the recombinantvariant enzymes obtained in Example 2-2, 50 mM Tris-HCl (pH 8.0), 3 mMMnSO₄, and each 5 mg/mL of the enzymes was added to 30% by weight offructose, and allowed to react at 60° C. for 2 hours. Furthermore, toevaluate thermal stability of the fructose-4-epimerization of theobtained recombinant variant enzymes, each 5 mg/mL of the enzymes wasleft at 60° C. for 3 hours, 24 hours, and 48 hours, and left on ice for5 minutes. Then, each of the heat-exposed variant enzymes was added at afinal concentration of 2 mg/ml to a substrate solution (50 mM Tris-HCl(pH 8.0) and 3 mM MnSO₄ were added to 30% by weight of fructose), andallowed to react at 60° C. for 2 hours.

As a result, all of the variants of the present disclosure had increasedfructose-4-epimerization stability, as compared with the wild-type (FIG.2 ). Further, the relative activity of some of the recombinant variantenzymes with respect to the wild-type was measured, and the results areshown in Table 4. As a result, it was found that their relative activitywas similar to or higher than that of the wild-type.

TABLE 4 Relative activity (%) KO 100 C52S 360 D136Y 98 T317Y 100 T317F100 L320F 98 S414E 97

The present inventors transformed into E. coli BL21(DE3) strain toprepare transformants (transformed microorganisms) designated as E. coliBL21(DE3)/CLKO_F4E_M4(D136F), E. coli BL21(DE3)/CLKO_F4E_M6(L320F), E.coli BL21(DE3)/CJ_KO_F4E_M7(S414E), respectively and deposited thetransformants on Sep. 19, 2018 at the Korean Culture Center ofMicroorganisms (KCCM) which is an International Depositary Authorityunder the provisions of the Budapest Treaty with Accession Nos.KCCM12323P (E. coli BL21(DE3)/CLKO_F4E_M4), KCCM12325P (E. coliBL21(DE3)/CLKO_F4E_M6), KCCM12326P (E. coli BL21(DE3)/CJ_KO_F4E_M7),respectively.

Based on the above description, it will be understood by those skilledin the art that the present disclosure may be implemented in a differentspecific form without changing the technical spirit or essentialcharacteristics thereof. Therefore, it should be understood that theabove embodiment is not limitative, but illustrative in all aspects. Thescope of the invention is defined by the appended claims rather than bythe description preceding them, and therefore all changes andmodifications that fall within metes and bounds of the claims, orequivalents of such metes and bounds are therefore intended to beembraced by the claims.

The invention claimed is:
 1. A fructose-4-epimerase variant having i)substitution of another amino acid for one or more amino acids selectedfrom the group consisting of amino acids at positions 52, 136, 197, 317,and 320, or substitution of glutamic acid (E) for an amino acid atposition 414 from the N-terminus of fructose-4-epimerase comprising theamino acid sequence of SEQ ID NO: 1, wherein the variant comprises anamino acid sequence having at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:
 1. 2. The fructose-4-epimerase variant ofclaim 1, wherein the another amino acid at position 52 is selected fromthe group consisting of methionine (M), threonine (T), serine (S), andleucine (L); the another amino acid for the amino acid at position 136is selected from the group consisting of phenylalanine (F), proline (P),tryptophan (W), and tyrosine (Y); the another amino acid for the aminoacid at position 197 is selected from the group consisting of alanine(A), and serine (S); the another amino acid for the amino acid atposition 317 is selected from the group consisting of tyrosine (Y), andphenylalanine (F); and the another amino acid for the amino acid atposition 320 is phenylalanine (F).
 3. A composition fir producingtagatose, the composition comprising the fructose-4-epimerase variant ofclaim
 1. 4. The composition for producing tagatose of claim 3, thecomposition further comprising fructose.
 5. A method of preparingtagatose, the method comprising the step of converting fructose intotagatose by contacting fructose with the fructose-4-epimerase variant ofclaim
 1. 6. A composition kw producing tagatose, the compositioncomprising the fructose-4-epimerase variant of claim
 2. 7. Thecomposition for producing tagatose of claim 6, the composition furthercomprising fructose.
 8. A method of preparing tagatose, the methodcomprising the step of converting fructose into tagatose by contactingfructose with the fructose-4-epimerase variant of claim 2.